Overview

In our project, we focus on precise treatment of liver cancer and have designed a modular mRNA therapy platform targeting liver cancer.

As a key part of this research, our team has developed a hardware solution aimed at advancing precise liver cancer treatment. As we learned from our research and own wet experiments, there are deficiencies in the accuracy and stability of drug delivery in current liver cancer treatment, which is an area urgently needing breakthroughs. We aim to create a tool that can enable our wet experiments to achieve higher protein expression levels, and more importantly, help realize efficient and precise delivery of RNA drugs to liver cancer cells, making the treatment more targeted and effective.

To achieve this goal, we have designed a microfluidic injection system that can precisely control the flow rates and mixing process of lipid phase and nucleic acid aqueous phase, thereby stably preparing and efficiently collecting high-quality lipid nanoparticle (LNP) carriers.

Meanwhile, combined with dynamic light scattering instrument and fluorescence spectrophotometer, we evaluate the particle size of LNPs and the fluorescence intensity of LNPs encapsulating fluorescently labeled nucleic acids respectively to verify the delivery effect of LNPs to cells.

Background

In the clinical and research fields of liver cancer treatment, RNA drugs have emerged as a crucial driver for advancing precise liver cancer therapy, thanks to their unique advantages such as precise regulation of abnormal gene expression in liver cancer cells and low toxic side effects. However, RNA molecules themselves have inherent characteristics: they are easily degraded by nucleases, highly hydrophilic, and hard to penetrate cell membranes. Without efficient delivery carriers, their therapeutic potential cannot be fully unleashed. Lipid nanoparticles (LNPs), by contrast, possess excellent biocompatibility, flexibly adjustable particle size, and tunable surface properties. They can effectively encapsulate RNA molecules to protect them from degradation, while enabling targeted delivery to liver cancer cells through surface modification. Therefore, the stable and efficient preparation of high-quality LNPs has become a core prerequisite for unlocking the application value of RNA drugs in liver cancer treatment, as well as a key breakthrough area urgently needed in the current research on precise liver cancer therapy.

Yet, from the perspective of the practical application of current LNP preparation technologies, two core issues severely hinder the progress of related research. On one hand, the traditional manual mixing method relies entirely on manual operation, making it impossible to precisely control the mixing ratio and rate of the lipid phase and nucleic acid aqueous phase. This leads to LNPs with a wide particle size distribution, significant fluctuations in the polydispersity index (PDI), and a high tendency for particle agglomeration, all of which fail to meet the strict requirements for LNP uniformity and stability in RNA drug delivery. On the other hand, commercial microfluidic platforms capable of high-quality LNP preparation on the market typically cost as much as $100,000, due to the integration of complex precision control modules, patented technologies, and supporting services. This exorbitant price creates an insurmountable barrier for academic laboratories and small research teams with limited funding, directly restricting in-depth exploration by numerous research teams in key areas such as LNP carrier optimization and RNA drug delivery efficiency improvement.

Design Rationale

In the team's early wet experiments, the aforementioned issues were more specific: preparing LNPs required operating two independent single-channel microfluidic syringe pumps (one for lipid phase, the other for nucleic acid aqueous phase) simultaneously. Operators had to switch between devices to adjust parameters like flow volume/rate separately and repeat synchronous calibration—making operations cumbersome and time-consuming. Worse, manual calibration often caused parameter asynchrony, leading to two-phase mixing ratio deviations that affected LNP particle size and encapsulation efficiency. Additionally, traditional microfluidic chips with simple channels failed to mix the two phases quickly and uniformly, worsening low LNP preparation efficiency and unstable quality; single experiments often needed repetitions to get qualified samples, increasing time and reagent costs.

Drawing on LNPs' core value in liver cancer precision therapy, industry pain points in current LNP preparation, and research teams' urgent need for low-cost, high-efficiency tools, the team developed an "integrated microfluidic injection system"—named PREMA (Precise Injection for Microfluidic LNP Assay). Integrating dual-channel syringe pump functions and a visual UI interface, PREMA enables centralized setting and synchronous adjustment of two-phase flow, eliminating device switching and ensuring parameter consistency. A custom microfluidic mixing chip (with optimized channels) was also developed for PREMA to boost mixing efficiency. The final PREMA not only solved wet experiment pain points (cumbersome operation, unstable quality) but also controlled costs to $100, only 1‰ of commercial equipment prices—by optimizing core components and simplifying non-essential modules.

This low-cost, high-performance system delivers significant research value: it breaks the "high-quality LNP preparation relies on expensive equipment" barrier, allowing cash-strapped teams to access stable LNP tools for cutting-edge research. Its user-friendly design also lowers microfluidic technology thresholds, even less experienced researchers can master it quickly, reducing operation-induced errors. This provides reliable hardware support for LNP-related research and drives efficiency and innovation in liver cancer precision therapy.

Mechanical Part of the Syringe Pump

All structural components of the PREMA's mechanical part are 3D-printed using PETG-CF, a material with excellent properties such as high strength, high rigidity, and fatigue resistance. The device uses an open-loop stepper motor as its power source, which drives the propulsion block via a lead screw and optical axis to achieve micron-level displacement control. Equipped with various fixing fixtures, it is compatible with syringes of different sizes, adapts to diverse microfluidic scenarios, and provides hardware support for the LNP preparation process.

Circuit Part of the Syringe Pump

An external power source connects via the power input interface, processed to power the entire circuit system. A power indicator light visually shows proper power connection and device startup, letting operators quickly check power status.

The WiFi module enables wireless communication with external devices and receives control commands from the browser UI interface, allowing remote and convenient control of the syringe pump.

Two stepper motor drive modules precisely control their respective stepper motors, enabling accurate adjustment of fluid flow rate, volume and other parameters in the syringe pump's dual channels to meet the demand for precise two-phase mixing control during LNP preparation.

These provide stroke limitation, preventing excessive movement of stepper motor-driven components. They protect the mechanical structure, avoid device damage from over-travel operation and ensure long-term stable operation of the syringe pump.

Equipment Iteration

Figure 4.1: Syringe Pump Version 1.0

Considering that the dual-channel design requires addressing the dual challenges of synchronized dual mechanical transmission and precise dual-parameter control, we prioritized the single-channel version to reduce development difficulty and ensure core function reliability. On one hand, the single-channel structure allowed repeated testing and optimization of the transmission accuracy of the stepper motor and lead screw-optical axis, verification of the adaptability of 3D-printed structural components, and confirmation of a stable mechanical link. On the other hand, we simultaneously debugged the control code to verify the accuracy of core programs such as flow rate adjustment and operating status feedback.

Figure 4.2: Syringe Pump Version 2.0

Building on the full verification of mechanical structure stability and code reliability from the single-channel device, we focused on key aspects during the subsequent development of the dual-channel version—including synchronized control of dual-channel parameters, and dual-channel parameter display and adjustment on the UI interface. Ultimately, we efficiently developed a dual-channel microfluidic syringe pump that meets experimental needs, ensuring stable and controllable performance across the entire chain from mechanics to control.

Microfluidic Chip Section

We independently completed the design and fabrication of the microfluidic chip. The process involves multiple strictly controlled steps: first, silicon wafer modification—cleaning with AB glue and treating with trimethylchlorosilane to create a high-quality substrate for subsequent PDMS molding; second, precisely preparing AB glue at a 10:1 mass ratio, fully stirring it, then performing glue pouring, bubble removal, and curing to form a stable AB glue structure; next, peeling, cutting, accurately drilling holes, and removing impurities; finally, bonding two PDMS sheets after plasma treatment and encapsulating them with high-temperature reinforcement.

The resulting microfluidic chip, when used with the self-developed syringe pump, enables efficient and uniform mixing of lipid and aqueous phases, providing strong support for high-quality LNP preparation.

Collection Device

We independently designed an integrated fixture to achieve efficient integration of the microfluidic chip and centrifuge tubes. Two clamping walls are arranged at the rear of the fixture: one for accurately collecting waste liquid during the preparation process, and the other for collecting the final prepared LNP product. This realizes an integrated "preparation-separation-collection" process, which not only simplifies operation steps but also avoids sample loss during transfer, improving the efficiency and integrity of LNP preparation. In addition, we customized the "DUT-China" team logo on the fixture surface via 3D printing. This design enhances the team recognition of the device and endows it with distinct team-specific characteristics.

Device Budget

Operation Method

In terms of operation and control design, the PREMA breaks through traditional limitations and subverts the mechanical control mode that relies on knobs and buttons. Equipped with a built-in WiFi module, the device eliminates the need for operators to connect additional dedicated controllers or install complex software. Instead, operators only need to connect any electronic device (such as a mobile phone, tablet, or computer) to the device's exclusive WiFi, then enter a specific domain name in the browser to quickly access the intuitive UI operation interface.

This interface allows direct setting of key parameters such as dual-channel flow rate, total volume, and operating time, while also displaying the pump's working status and data in real time—making the entire operation process simple and efficient, and significantly lowering the usage threshold. Compared with the cumbersome operation of traditional syringe pumps (which require close-range manual knob adjustment and repeated parameter calibration), this wireless, web-based control design not only makes operation more flexible and convenient, but also better adapts to laboratory scenarios such as multi-device collaboration and remote monitoring, further improving the operational efficiency and flexibility of the LNP preparation process.

Parameter Comparison

To further verify the performance of the PREMA, we selected the Longer LSP01 - 3A syringe pump as a control.

Testing

To comprehensively evaluate the actual performance of the PREMA in lipid nanoparticle (LNP) preparation, we conducted rigorous comparative experiments where the Longer syringe pump and the traditional manual mixing method were selected as controls, with analysis focused on two key indicators: particle size and fluorescence intensity; during the experiments, we strictly followed standardized operating procedures to ensure consistent conditions across the three groups, including lipid material formulation, nucleic acid concentration, and reaction temperature, and a dynamic light scattering (DLS) instrument was used for accurate measurement of LNP particle size while a fluorescence spectrophotometer determined the fluorescence intensity of LNPs encapsulating fluorescently labeled nucleic acids.

For the evaluation of LNP quality based on particle size and polydispersity index (PDI), LNPs prepared by the self-developed and Longer syringe pumps showed relatively concentrated particle size ranges and good uniformity, while LNPs from manual mixing had a wide, irregular particle size distribution with significant size differences; in terms of PDI, while the PDI of LNPs from the self-developed and Longer pumps fluctuated slightly, their overall particle size distribution remained consistent, and LNPs from manual mixing exhibited extreme PDI fluctuations, resulting in highly unstable particle size uniformity. In summary, both the self-developed and Longer syringe pumps stably produce LNPs with particle sizes of 50-150nm and good uniformity, but the manual method leads to large particle size errors, obvious particle agglomeration, and uneven sizes; although the self-developed pump lags slightly behind the Longer pump in PDI, it has advantages in functions like dual-channel continuous preparation, and LNP uniformity can be further improved by optimizing processes.

For fluorescence intensity analysis, LNPs prepared by the self-developed and Longer syringe pumps showed similar, high fluorescence intensities, indicating effective encapsulation of fluorescently labeled nucleic acids, while the manual mixing method produced LNPs with significantly lower fluorescence intensity, meaning a large amount of nucleic acid remained unencapsulated (in a free state)—this would greatly reduce the delivery efficiency of nucleic acid drugs in subsequent LNP applications. Combining the above results, the self-developed microfluidic dual-channel syringe pump demonstrates performance comparable to the mature Longer brand in LNP preparation, enabling stable, efficient production of high-quality LNPs at a lower cost, and the traditional manual mixing method performs poorly in key LNP preparation indicators, failing to meet precise, efficient requirements—further highlighting the application value and potential of the PREMA in LNP preparation;

Currently, the microfluidic injection system can only complete one round of LNP preparation with a single syringe loading, and manual refilling is required for subsequent batches; looking ahead, we aim to upgrade the device to a continuous production system, which will involve optimizing the fluid supply module to enable automatic, continuous liquid replenishment, eliminating the need for manual intervention between preparation cycles; by achieving continuous liquid feeding and LNP preparation, we can significantly improve LNP production efficiency, making the system more suitable for large-scale or high-throughput LNP preparation scenarios and further expanding its application potential in biomedical research and related fields.

Reference

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